Efficient translocation across biological membranes is crucial for the delivery of therapeutic and diagnostic agents. In addition to drug delivery, there are many potential in vitro applications in areas such as drug discovery and laboratory assays that could benefit from improved intracellular delivery of biomolecules and macromolecular cargo. Protein transduction domains (PTDs; also referred to as cell penetrating peptides) have attracted considerable interest in the drug delivery field for their ability to translocate across biological membranes. The PTDs are relatively short sequences that confer this apparent translocation activity to proteins and other macromolecular cargo to which they are conjugated, complexed or fused (Derossi et al. (1994) J. Biol. Chem. 269:10444-50; Fawell et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:664-68; Elliott & O'Hare (1997) Cell 88:223-33; Schwarze et al. (2000) Trends Cell Biol. 10:290-5; Snyder & Dowdy (2001) Curr. Opin. Mol. Ther. 3:147-52; Bennett et al. (2002) Nat. Biotechnol. 20:20). The translocation mechanism is still unclear, but studies with PTDs have suggested that translocation is an energy-independent and nonreceptor-mediated event and that most cell types can be targeted (Derossi et al. (1994) J. Biol. Chem. 269:10444-50; Derossi et al. (1996) J. Biol. Chem. 271:18188-93; Vives et al. (1997) J. Biol. Chem. 272:16010-7; Nagahara et al. (1998) Nat. Med. 4:1449-52; Schwarze et al. (2000) Trends Cell Biol. 10:290-5). However, some recent studies have suggested that the PTDs direct highly efficient cellular uptake through regular pinocytotic mechanisms (Leifert et al. (2002) Gene Ther. 9:1422-8; Fittipaldi et al. (2003) J. Biol. Chem. 278:34141-9; Leifert & Lindsay Whitton (2003) Mol. Ther. 8:13-20; Lundberg et al. (2003) Mol. Ther. 8:143-50; Richard et al. (2003) J. Biol. Chem. 278:585-90; Vives et al. (2003) Curr. Protein Pept. Sci. 4:125-32).
Linkage of PTDs to molecules of interest has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For the delivery of protein cargo, for example, the protein may be covalently linked to a PTD in a fusion protein. There is a need in the art for methods and compositions that facilitate the linkage of PTDs to cargo. The present invention addresses this and other needs.